KR20090072170A - Vesicle immobilizing glucose oxidase and method for production of it - Google Patents

Abstract

A vesicle which glucose oxidase (GOD) is fixed is provided to deliver target material in an expected concentration without loss of blood and solution by selectively crushing the vesicle in solution containing blood or glucose. A vesicle which has a spherical form by amphipathic molecule and contains a target material inside comprises a glucose oxidase fixed on the outer surface. The amphipathic molecule is dioleoylphosphatidylethanolamine(DOPE). The hydrophobic anchor is palmitic acid N-hydroxysuccinimide ester. A method for manufacturing the vesicle comprises: a step of producing suspension after adding amphipathic molecule in a solution containing target material and dispersion agent; a step of homogenizing suspension solution with sonication, microfluidizer or membrane extrusion; and a step of removing the dispersion agent using gel chromatography or dialysis.

Description

Vesicles to which glucose oxidase is immobilized and a method for preparing the same {Vesicle immobilizing glucose oxidase and method for production of it}

The present invention relates to a vesicle which is formed in a spherical shape by both lipophilic molecules and a target material can be collected therein, and more particularly, a glucose oxidase (GOD) is formed on an outer surface thereof. It relates to a vesicle immobilized on and to a method of manufacturing the same.

Amphiphilic molecules with a packing parameter close to 1 form bilayer vesicles, or vesicles, in the water phase.

A vesicle is a closed spherical endoplasmic reticulum formed by a bilayer membrane with hydrophilic groups facing the water phase and facing hydrophobic groups.

Since the vesicle consists of an inner water core and a hydrophobic bilayer membrane, it is possible to enclose both hydrophobic and hydrophilic drugs at the same time. In addition, the release rate of the drug is gradually released, and the rate of release can be controlled by adjusting the composition of the vesicle. Especially, the vesicle (liposome) composed of phospholipids exhibits biocompatibility, so the drug in the human body Research into developing a drug carrier has been actively conducted recently (Kim JC, Song ME, Kim MJ, Lee EJ, Park SK, Rang MJ, Ahn HJ (2002) Preparation and characterization of Triclosan-containing vesicles). Colloid Surf.B: Biointerfaces, 26: 235-241).

As such, vesicles are used as mediators to capture and deliver drugs and other active ingredients (hereinafter collectively referred to as 'target substances') to the human body. If possible, there is an advantage that can maximize the utility of the target material collected inside.

Accordingly, an object of the present invention is to prepare a vesicle which can be selectively crushed, and in particular, to produce a vesicle that can be selectively crushed by blood glucose concentration.

In order to achieve the above object, the present invention is spherical formed by both lipophilic molecules, and in the vesicle which can collect the target substance therein, glucose oxidase is immobilized on the outer surface of the vesicle. Provide a vesicle.

In addition, the present invention comprises the steps of preparing a suspension by adding both lipophilic molecules to an aqueous solution in which the hydrophobized glucose oxidase, the target substance collected in the vesicle and the dispersant are dissolved, and then hydrated to prepare a suspension; Homogenizing the suspension (b); And after homogenization, the step of removing the dispersant (c); provides a method for producing a vesicle comprising a.

Hereinafter, the content of the present invention will be described in more detail.

The present invention provides a vesicle formed spherically by both lipophilic molecules and characterized in that glucose oxidase is immobilized on the outer surface of the vesicle in a vesicle in which a target substance can be collected therein.

Glucose oxidase (GOD) immobilized on the outer surface of the vesicle of the present invention oxidizes glucose in the blood when the vesicle is injected into the blood to produce gluconic acid. By the production of gluconic acid, the pH around the vesicle is lowered to become acidic. As a result, the vesicle is destroyed and the target substance enclosed therein is released (FIG. 1).

Therefore, the vesicle of the present invention is selectively crushed by the concentration of glucose in the blood, thereby exhibiting the advantage of being able to deliver the target substance significantly appropriately.

Becyclo immobilization of glucose oxidase can be carried out through various immobilization methods via covalent and non-covalent bonds, preferably by attaching a hydrophobic anchor to the glucose oxidase, thereby providing a hydrophobic bond to the inner hydrophobic portion of the vesicle membrane. It can be used to immobilize via.

Hydrophobic anchors can bind to glucose oxidase and impart hydrophobic properties to glucose oxidase, so that various compounds including amino acids having hydrophobic residues can be used without being limited to specific ones. For example, palmitic acid Palmitic acid N-hydroxysuccinimide ester can be used.

Meanwhile, both lipophilic molecules capable of forming the vesicle of the present invention have both hydrophilic and hydrophobic groups in the molecule. When suspended in a solution, a bispherical vesicle closed by forming a bilayer membrane. It is not limited to a specific material as long as it can form, but both lipophilic molecules having a packing parameter close to 1 having almost the same cross-sectional area of the hydrophilic head and the hydrophobic tail are easy to form spherical vesicles. It is preferable because of that.

On the other hand, dioleoylphosphatidylethanolamine (DOPE) in the phospholipids, both amphiphilic molecules, has a molecular shape because of its packing parameter greater than 1, that is, its cross-sectional area of the hydrophilic head is smaller than that of the hydrophobic tail. )to be. Thus, DOPE does not form a bilayer vesicle in the water phase and forms an inverted hexagonal phase. However, if a suitable complementary molecule is present, the double molecule can form a double layer membrane, since the auxiliary molecule fills the space between the hydrophilic head group and the head of the DOPE. Auxiliary molecules capable of forming a bilayer membrane include (dinitro-phenyl) caproyl-phosphatidyl-ethanolamine, N-succinyldioleoyl-phosphatidyl-ethanolamine (N -succinyldioleoyl-phosphatidyl-ethanolamine), ganglioside, oleic acid, cholesteryl hemisuccinate, palmitoyl homocystein, glycophorin A , Palmitoyl immunoglobulin, and palmitoyl lysozyme have been found (Kim Jin-cheol, Kim Jong-deok, Recent trends in liposome use, Recent advances in Bioprocess Engineering, 4, 65-83, 1996).

However, in the present invention, glucose oxidase (GOD) is preferably hydrophobicized and immobilized on vesicles, which acts as an auxiliary molecule in vesicle formation, and plays a large role in preparing liposomes (vesicles) from DOPE. . In other words, hydrophobized glucose oxidase (HMGOD) serves to oxidize glucose and to serve as an auxiliary molecule to help the formation of vesicles.

On the other hand, the present invention comprises the steps of hydrating glucose oxidase, the target material collected in the vesicle and the dispersing agent is dissolved in the aqueous solution in which both lipophilic molecules are added and then hydrated to prepare a suspension; Homogenizing the suspension (b); And after homogenization, the step of removing the dispersant (c); provides a method for producing a vesicle comprising a.

At this time, preferably, both lipophilic molecules are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylacid (PA), phosphatidylacid (PA) Phospholipids such as inositol (PI), dioleoylphosphatidylethanolamine (DOPE); Ester compounds such as mono glycerides, di glycerides and tri glycerides; Fatty acids, including palmitic acid, stearic acid, behenic acid; Alkyl amines including dimethylaminopropyloctadecanamide, dodecyl amine, myristyl amine, palmityl amine, stearyl amine ; And alkyl alcohols including dodecyl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol; It is good to be any one selected from among.

On the other hand, in the vesicle manufacturing method of the present invention, if the target material can be collected inside the vesicle is not necessarily limited to a specific one, preferably insulin (insulin), glipizide, repagridge Hypoglycemic agents such as regaglizinide, metformin, pioglitazone, doxorubicin, paclitaxel, gemcitabine, transcizumab, transtuzumab, cisplatin zecilatin anticancer drugs such as gemeracil, leucovorin, and ampicillin, amoxicillin, moxalactam, roxithromycin, doxycycline, levofloxacin Antibiotics and the like can be used.

On the other hand, in the vesicle manufacturing method of the present invention, the concentration of the dispersant is 0.01 to 0.1% (w / w), preferably 0.05 to 0.5% (w / w), most preferably 0.07% to 0.12% (w / w) is good. If it is higher than the range of 0.01 to 0.1% (w / w), the hydrophobized glucose oxidase is denatured and if it is lower than this range, the dry phospholipid membrane cannot be effectively dispersed.

At this time, as the dispersant, it is preferable to use a surfactant having a hydrophilic liphophilic balance number (HLB no.) Of 4 to 15, more preferably 6 to 14, and most preferably 7 to 13. good. If it is higher than the range of 4-15, glucose oxidase is denatured, and if it is lower than this range, the dry phospholipid membrane cannot be effectively dispersed. Surfactants having a range of these values include polyhydric alcohol esters such as sorbitan laurate, sorbitan palmitate, and PEO (20) sorbitan trioleate (PEO (20) sorbitan trioleate). Ethylene oxide adducts such as ethylene oxide, polyhydric alcohol esters, and bile salts such as deoxycholate may be used.

On the other hand, the concentration of the hydrophobized glucose oxidase in the vesicle preparation method of the present invention is preferably 1000: 1 to 1,000,000, more preferably 5000: 1 to 500,000: 1 as a molar ratio to both lipophilic molecules. , Most preferably, 10000: 1 to 100,000: 1. At rates outside the 1000: 1 to 1,000,000 range, the vesicles do not form or do not act as vesicles as drug carriers.

On the other hand, in the vesicle manufacturing method of the present invention, the homogenization of the suspension is not necessarily limited to a specific homogenization method, but preferably ultrasonic sonication, microfluidizer, and membrane extrusion method. It is good to use any one selected from).

In the vesicle production method of the present invention, the removal of the dispersant is not necessarily limited to a specific method, but gel chromatography or dialysis is preferably used. When performing gel chromatography, Sephadex, Biogel, etc. are preferably used as the filling gel.When using the dialysis method, the volume ratio of the dialysate to the suspension is at least 1: 100, and the dialysis is performed. The time should be 24 hours, and the dialysis solution should be changed until the dispersant and the unsealed target substance are completely removed.

Meanwhile, in the vesicle manufacturing method of the present invention, the hydrophobized glucose oxidase (HMGOD) is not limited to one prepared by a specific method, but preferably, dissolving the hydrophobic anchor in an organic solvent (A); Slowly adding the organic solution in which the hydrophobic anchor is dissolved to the aqueous solution in which the dispersant and glucose oxidase are dissolved (b); Recovering the filtrate by filtration after the reaction is completed (c); And (d) removing the dispersant from the recovered filtrate.

In this case, the hydrophobic anchor is not necessarily limited to a specific one, and palmitic acid N-hydroxysuccinimide ester (PAHE) may be used as an example.

On the other hand, in the method for preparing hydrophobized glucose oxidase (HMGOD) of the present invention, the concentration of the dispersant in the aqueous solution is preferably 0.1 to 5.0% (w / w), more preferably 0.5 to 4.0% (w / w) Most preferably, it is 1.0 to 3.0% (w / w). If it is outside the range of 0.1 to 5.0% (w / w), glucose oxidase is denatured or hydrophobic anchor cannot be effectively dispersed.

In this case, the dispersant may be a surfactant having a hydrophilic liphophilic balance number (HLB no.) Of preferably 4 to 15, more preferably 6 to 14, and most preferably 7 to 13. . If it is higher than the range of 4-15, GOD is denatured, and if it is lower than this range, hydrophobic anchors cannot be effectively dispersed. Surfactants having a range of these values include polyhydric alcohol esters such as sorbitan laurate, sorbitan palmitate, and PEO (20) sorbitan trioleate (PEO (20) sorbitan trioleate). Ethylene oxide adducts such as ethylene oxide, polyhydric alcohol esters, and bile salts such as deoxycholate may be used.

On the other hand, in the method for producing hydrophobized glucose oxidase (HMGOD) of the present invention, the concentration of glucose oxidase (GOD) in the aqueous solution is preferably 0.1 to 1.0% (w / w), more preferably 0.2 to 0.8% ( w / w), most preferably 0.3 to 0.6% (w / w). This is because the reaction does not proceed if it is outside the range of 0.1 to 1.0% (w / w).

On the other hand, in the method for producing hydrophobized glucose oxidase (HMGOD) of the present invention, the concentration of the hydrophobic anchor is preferably 0.3 to 3.0% (w / w), more preferably 0.5 to 2.0% (w / w) in the organic solution. Most preferably, it is 0.7 to 1.5% (w / w). When using concentrations lower than 0.3 to 3.0% (w / w), glucose oxidase is added because a large amount of organic solvent is added to the aqueous phase in order to add the required amount of hydrophobic anchor (eg PAHE) to the aqueous phase. This is because it may be denatured, and if a concentration higher than 3 to 3.0% (w / w) is used, the hydrophobic anchor may not be dissolved or aggregated so that the reaction does not proceed.

Meanwhile, in the method for preparing hydrophobized glucose oxidase (HMGOD) of the present invention, the molar ratio of the hydrophobic anchor (eg, PAHE) to the glucose oxidase is preferably 1: 200 to 1: 10, more preferably 1: 100 to 1:20, most preferably 1:50 to 1:30. If the ratio is lower than the ratio of 1: 200 to 1:10, the hydrophobic alkyl group is less covalently bonded to the GOD. Therefore, the hydrophobic alkyl group cannot be immobilized on the surface of the vesicle (liposome). This is because excessively covalently binds to lowers the enzyme activity of GOD.

On the other hand, in order to completely remove the dispersant in the hydrophobized glucose oxidase (HMGOD) production method of the present invention, it is preferable to use the method of dialysis, the volume ratio of the dialysate to the filtrate is at least 1: 100 , Dialysis time is 12 hours, it is good to repeat three times while changing the dialysate.

Since the vesicle of the present invention is immobilized on the outer surface of the glucose oxidase, when the vesicle is injected into the blood or the solution containing glucose, the glucose in the blood or the solution is oxidized to produce gluconic acid. By the production of gluconic acid, the pH around the vesicle is lowered to become acidic, and the vesicle is destroyed. At this time, the target substance enclosed therein is released to the outside.

Accordingly, the vesicles of the present invention can be selectively crushed in a blood or glucose-containing solution, thereby exerting the advantage of delivering a target substance at a predetermined concentration without loss in blood or solution, which is very useful in the pharmaceutical and food industries.

Hereinafter, the content of the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, but includes modifications of equivalent technical ideas.

Example  1: hydrophobized Glucose  Oxidase ( HMGOD)  Produce

First, 0.24 g of surfactant deoxycholate (DOC) was dissolved in 12 ml of phosphate buffer (pH 8.8, 0.16M). Then 0.048 g of glucose oxidase (GOD) was dissolved in the previously prepared aqueous solution.

On the other hand, 0.0228 g of palmitic acid N-hydroxysuccinimide ester was dissolved in 2 ml of dioxane, an organic solvent. Thereafter, 0.4 ml of the organic solution in which the ester was dissolved was slowly added to the aqueous solution in which GOD was dissolved. At this time, the molar ratio of GOD to palmitic acid ester was 1:40. The reaction proceeded for 10 hours with stirring at 30 ° C.

After the reaction, it was filtered through a filter with 0.45 μm pores to remove byproducts of the reaction. Then, the filtrate was placed in a dialysis membrane to remove dioxycholate and low molecular impurities, and dialyzed in 1000 ml phosphate buffer (pH 7.4, 0.15 M) for 12 hours. At this time, dialysis was repeated three times to completely remove the low molecular impurities. The dialysis solution was then lyophilized to obtain dry hydrophobized glucose oxidase (HMGOD).

Evaluation example  One: Glucose  Oxidase ( GOD To) Combined Palmitic  Acid palmitic  determination of the number of acids)

The number of palmitic acid covalently bound to GOD was determined using the TNBS method.

First, 0.025 g sodium lauryl sulfate was dissolved in 50 ml phosphate buffer (pH 8.9, 0.01 M).

Meanwhile, trinitrobenzene sulphonic acid (TNBS) solution (0.03%) was prepared by adding 0.5 ml trinitrobenzene sulphonic acid to the 83 ml sodium lauryl sulfate aqueous solution prepared above. Then, 1 ml sodium lauryl sulfate aqueous solution, 1 ml TNBS solution, and 0.5 ml HMGOD aqueous solution (3.55 mg / ml) were placed in a 10 ml glass container and reacted at 50 ° C. for 1 hour. Then 1.9 ml HCl aqueous solution (0.21 M) was added to the reaction mixture and left at room temperature for 30 minutes.

Thereafter, the number of amino groups was determined by measuring the absorbance of the reactants at 335 nm. The difference between the number of amino groups in GOD and the number of amino groups in HMGOD is the number of covalent bonds of palmitic acid. According to the experimental results, the number of amino groups in one GOD molecule was 24 ± 1 and the number of amino groups in HMGOD was 18 ± 1. .

Therefore, it can be said that 5 ± 1 amino group is covalently bonded to each GOD molecule.

Example 2 and Comparative example  1 to 3: Glucose  Oxidase ( GOD ) Is fixed Vesicle  Produce

                                                        (Mol%) DPPC DOPE HMGOD DOC Comparative Example 1 86% - - 14% Comparative Example 2 85.997% - 0.003% 14% Comparative Example 3 - 86% - 14% Example 2 - 85.997% 0.003% 14%

The vesicles were prepared according to the composition and the method described in the detailed description of the invention as in the above table. As in Comparative Examples 1 and 2, when DPPC (Dipalmitoylphosphatidylcholine) was used as the amphiphilic molecule, vesicles were formed regardless of the presence or absence of HMGOD. This is because the molecular shape of the DPPC is a rectangle whose cross-sectional area of the hydrophilic head and the hydrophobic tail are similar. Thus, bilayer lipid membranes could be formed regardless of the presence of HMGOD.

On the other hand, when using DOPE as a lipid as in Example 2 and Comparative Example 3, the vesicle is formed when HMGOD is present, the vesicle was not formed if not present. In the case of DOPE, the head is too small and triangular. Therefore, a vesicle is formed only when an auxiliary molecule such as HMGOD is present, or when the vesicle is not formed.

Evaluation example  2: present invention Vesicle  Evaluation of Active Component Release Behavior in Glucose Solution

Using a fluorescent substance calcein (calcein) as a model material by the method of the present invention, by preparing a vesicle in which glucose oxidase is inserted according to the composition as described in Table 2 below, the release experiment was carried out under the conditions of various glucose concentrations. Was carried out. Distilled water was adjusted to pH 7.5 using NaOH or HCl solution and glucose was added so that each glucose concentration was 0, 50, 200, 400 mg / dL. The vesicle suspension was added thereto and the fluorescence of calcein was measured for 3 hours while stirring using a magnetic bar (Hitachi F-2500, Japan).

division Amount of Release Solution (mL) Amount of liposome suspension (mL) Glucose concentration (mg / dL) Example 3 50 1.724 0 Example 4 50 1.724 50 Example 5 50 1.724 200 Example 6 50 1.724 400

As a result, the vesicle release in the example with high glucose concentration was higher than that in the example with low glucose concentration (FIG. 2). In Example 4 (○), liposome release was about 20% for 1 hour, 40% in Example 5 (▼), and 60% in Example 6 (▽).

In addition, in the release after 3 hours, about 80% was released in Example 4, but in Example 5, 6, about 95% was released. However, liposome release in Example 3 (control,?), Which was in the absence of glucose, was hardly achieved for 3 hours.

From the above experiments, it was confirmed that the vesicle of the present invention was selectively crushed in the glucose-containing solution, and the target substance collected therein was released into the solution.

1 is a schematic diagram of a vesicle to which glucose oxidase is immobilized.

2 is a graph evaluating the active ingredient release behavior in the glucose solution of the vesicle of the present invention.

Claims (18)

In a vesicle formed spherically by both lipophilic molecules and the target material can be collected therein, Vesicles characterized in that glucose oxidase is immobilized on the outer surface of the vesicle The method of claim 1, Glucose oxidase, A vesicle characterized by having a hydrophobic anchor attached thereto The method of claim 2, Both lipophilic molecules, A vesicle characterized by a dioleoylphosphatidylethanolamine (DOPE) The method of claim 2, Hydrophobic anchors, A vesicle characterized by a palmitic acid hydroxysuccinimide ester (A) preparing a suspension by adding both lipophilic molecules to an aqueous solution in which a hydrophobized glucose oxidase, a target substance collected in a vesicle, and a dispersant are dissolved; Homogenizing the suspension (b); And After homogenization, the step of removing the dispersant (c); method of producing a vesicle comprising a The method of claim 5, Both lipophilic molecules, Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylacid (PA), phosphatidylinositol (PI), phosphatidylinositol; Phosphorus lipids such as dioleoylphosphatidylethanolamine (DOPE); Ester compounds such as mono glycerides, di glycerides and tri glycerides; Fatty acids, including palmitic acid, stearic acid, behenic acid; Alkyl amines including dimethylaminopropyloctadecanamide, dodecyl amine, myristyl amine, palmityl amine, stearyl amine ; And, Alkyl alcohols, including dodecyl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol; Method for producing a vesicle, characterized in that any one selected from The method of claim 5, The concentration of the dispersant is Method for producing a vesicle, characterized in that 0.01 to 0.1% (w / w) in the aqueous solution The method of claim 7, wherein Dispersant, Method for producing vesicles characterized in that the hydrophilic liphophilic balance number (HLB no.) Is a surfactant of 4 to 15 The method of claim 5, The concentration of hydrophobized glucose oxidase is Method for producing a vesicle, characterized in that the molar ratio for both lipophilic molecules, 1000: 1 to 1,000,000 The method of claim 5, Homogenization is Method for producing a vesicle characterized in that any one of ultrasonication, microfluidizer and membrane extrusion method is used. The method of claim 5, Removal of dispersant, Method for producing a vesicle, characterized in that using gel chromatography or dialysis The method of claim 5, Hydrophobized glucose oxidase, Dissolving the hydrophobic anchor in an organic solvent (A); Slowly adding the organic solution in which the hydrophobic anchor is dissolved to the aqueous solution in which the dispersant and glucose oxidase are dissolved (b); Recovering the filtrate by filtration after the reaction is completed (c); And Removing the dispersant from the recovered filtrate (D); method for producing a vesicle characterized in that it was prepared through the process comprising The method of claim 12, Hydrophobic anchors, Method for producing a vesicle characterized in that the palmitic acid hydroxysuccinimide ester (palmitic acid N-hydroxysuccinimide ester) The method of claim 12, The concentration of the dispersant is Method for producing a vesicle, characterized in that 0.01 to 5.0% (w / w) in the aqueous solution The method of claim 14, Dispersant, Method for producing vesicles characterized in that the hydrophilic liphophilic balance number (HLB no.) Is a surfactant of 4 to 15 The method of claim 12, Glucose oxidase, Method for producing a vesicle, characterized in that 0.1 to 1.0% (w / w) in an aqueous solution The method of claim 12, Hydrophobic anchors, Method for producing a vesicle, characterized in that 0.3 ~ 3.0% (w / w) in the organic solution The method of claim 12, The addition ratio of the organic solution and the aqueous solution of step (b) is Method for producing a vesicle characterized in that the molar ratio of the hydrophobic anchor to glucose oxidase is 1: 200 ~ 1:10 added

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